Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Abstract

Rectal gland tubule (RGT) segements of the spiny dogfish (Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PD_te resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of this membrane (FR_bl), and intracellular activities of Na⁺, Cl⁻, K⁺ (a ^cell) were examined. Furosemide (5·10⁻⁴ mol·l⁻¹) reduced PD_te from −12±0.7 to −2.3±0.2 mV (n=63), hyperpolarized PD_bl from −71±1.3 to −79±0.9 mV (n=59), FR_bl decreased from 0.2±0.03 to 0.13±0.01 (n=21),a ^cell_Cl− fell from 38±4 to 11±2 mmol·l⁻¹ (n=14),a ^cell_Na+ fell from 37±4 to 17±2 mmol·l⁻¹ (n=12) anda ^cell_K+ was constant [113±14 vs. 117±15 mmol·l⁻¹ (n=6)]. Furosemide exerted its effects within some 20–40 s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall ina ^cell_Cl− was approximately twice as rapid when compared to that ofa ^cell_Na+ . Ba²⁺ 0.5 mmol·l⁻¹ (bath) reduced PD_te from −7.4±1.2 to −4.1±0.6 mV (n=24), increased R_te from 18±2 to 22±2.5, Ωcm² (n=14). PD_bl depolarized from −75±2 to −48±2 mV (n=42), FR_bl increased from 0.2±0.02 to 0.34±0.04 (n=14) anda ^cell_K increased from 143±28 to 188±40 mmol·l⁻¹ (n=4). Ouabain (50·10⁻⁶ mol·l⁻¹, bath) reduced PD_te from −12±2 to −3±0.5 mV (n=9), R_te increased from 18±3 to 21±3 Ωcm² (n=5), PD_bl depolarized from −67±4 to −26±3 mV (n=14), FR_bl increased from 0.23±0.04 to 0.45±0.05 (n=6),a ^cell_K fell only slightly from 135±15 to 112±30 mmol·l⁻¹ (n=4), buta ^cell_Cl− increased from 35±12 to 111±37 mmol·l⁻¹ (n=3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba²⁺. The ouabain effects on PD_te and PD_bl were completely prevented if furosemide was applied first. Amiloride (≤10⁻³ mol·l⁻¹, both sides), and anthracene-9-carboxylate (≤10⁻³ mol·l⁻¹, lumen) were devoid of effects on PD_te and PD_bl. The stilbene disulfonate derivate SITS (≤10⁻³ mol·l⁻¹, both sides) led to a 36±9% inhibition of PD_te. The present data, apart from supporting the cell model proposed in a preceding report, allow the following conclusions: The transient analysis of the furosemide effects is suggestive for a 2 Cl⁻∶1 Na⁺ stoichiometry of the carrier. The furosemide induced reduction in FR_bl suggests that the fall ina ^cell_Cl− leads to a marked reduction in the apical Cl⁻-conductance. Furosemide apparently drivesa Cl ⁻/cell to passive distribution and PD_bl to the K⁺ equilibrium potential.a ^cell_Na+ then approaches a minimal value which is close to that present in nonstimulated RGT segments. Ba²⁺ leads to an increase ina ^cell_K+ because it blocks the conductive recycling pathway for this ion. Ouabain does not only inhibit the (Na⁺+K⁺)-ATPase, it also reduces the K⁺-conductance of the basolateral membrane. This explains whya ^cell_K+ stays high. The cell PD then is dominated bya ^cell_Cl− . The Na⁺ 2Cl⁻K⁺-carrier increasesa ^cell_Cl− further and the cell continues to depolarize. Thus, the depolarization of the cell is not a sign of K⁺ loss but indicative of Cl⁻ gain. In the presence of furosemide ouabain is devoid of any effect on PD_bl, suggesting that the Na⁺2 Cl⁻K⁺-carrier is the only quantitatively important source of Na⁺ entry into the RGT cell.